G Protein-coupled Receptor Kinase 2 Upregulation Research Articles

Overexpression of GRK2 in vascular smooth muscle leads to inappropriate hypertension and acute heart failure as in clinical scenario 1

Clinical scenario 1 (CS1) is acute heart failure (HF) characterized by transient systolic blood pressure (SBP) elevation and pulmonary congestion. Although it is managed by vasodilators, the molecular mechanism remains unclear. The sympathetic nervous system plays a key role in HF, and desensitization of cardiac β-adrenergic receptor (AR) signaling due to G protein-coupled receptor kinase 2 (GRK2) upregulation is known. However, vascular β-AR signaling that regulates cardiac afterload remains unelucidated in HF. We hypothesized that upregulation of vascular GRK2 leads to pathological conditions similar to CS1. GRK2 was overexpressed in vascular smooth muscle (VSM) of normal adult male mice by peritoneally injected adeno-associated viral vectors driven by the myosin heavy chain 11 promoter. Upregulation of GRK2 in VSM of GRK2 overexpressing mice augmented the absolute increase in SBP (+ 22.5 ± 4.3 mmHg vs. + 36.0 ± 4.0 mmHg, P < 0.01) and lung wet weight (4.28 ± 0.05 mg/g vs. 4.76 ± 0.15 mg/g, P < 0.01) by epinephrine as compared to those in control mice. Additionally, the expression of brain natriuretic peptide mRNA was doubled in GRK2 overexpressing mice as compared to that in control mice (P < 0.05). These findings were similar to CS1. GRK2 overexpression in VSM may cause inappropriate hypertension and HF, as in CS1.

Myocardial GRK2 Reduces Fatty Acid Metabolism and β-Adrenergic Receptor-Mediated Mitochondrial Responses.

G-protein coupled receptor (GPCR) kinase 2 (GRK2) is upregulated in heart failure (HF) patients and mouse models of cardiac disease. GRK2 is a regulator of β-adrenergic receptors (βARs), a GPCR involved in ionotropic and chronotropic responses. We and others have recently reported GRK2 to be localized in the mitochondria, although its function in the mitochondria and/or metabolism remain not clearly defined. We hypothesized that upregulation of GRK2 reduced mitochondrial respiratory function and responses to βAR activation. Utilizing isolated mouse primary adult cardiomyocytes (ACMs), we investigated the role of glucose, palmitate, ketone bodies, and BCAAs in mediating cell survival. Our results showed that myocyte upregulation of GRK2 promotes palmitate-induced cell death. Isotopologue labeling and mass spectrometry showed that the upregulation of GRK2 reduces β-hydroxybutyryl CoA generation. Next, using isoproterenol (ISO), a non-selective βAR-agonist, we determined mitochondrial function in mouse and human primary ACMs. Upregulation of GRK2 impaired ISO-mediated mitochondrial functional responses, which we propose is important for metabolic adaptations in pathological conditions. Increased cardiac levels of GRK2 reduced fatty acid-specific catabolic pathways and impaired ISO-stimulated mitochondrial function. Our data support the notion that GRK2 participates in bioenergetic remodeling and may be an important avenue for the development of novel pharmacological strategies in HF.

Lycium barbarum polysaccharide antagonizes cardiomyocyte apoptosis by inhibiting the upregulation of GRK2 induced by I/R injury, and salvage mitochondrial fission/fusion imbalance and AKT/eNOS signaling

Ischemia–reperfusion (I/R) injury is the main reason why infarct size continues to progress during the process of restoring myocardial perfusion, and it significantly increases the risk of death. At present, the therapeutic effects of clinically used drugs are limited. Therefore, it is particularly necessary to explore myocardial-protective agents that effectively prevent I/R injury. Lycium barbarum polysaccharide (LBP) is a water-soluble polysaccharide extracted from wolfberry fruit. In this study, we found that LBP limited myocardial infarct size, improved adverse remodeling, and reduced cell death and oxidative stress. G protein-coupled receptor kinase-2 (GRK2) is a key molecule involved in myocardial I/R injury. In vivo and in vitro experiments showed that LBP inhibited the upregulation of GRK2 expression induced by I/R injury, which was related to the antiapoptotic effect of LBP. In addition, we found that LBP partially restored I/R-induced mitochondrial fission/fusion imbalance, as well as levels of phosphorylated protein kinase B (p-AKT) and phosphorylated endothelial cell nitric oxide synthase (p-eNOS), and this restorative effect could be attenuated by overexpression of GRK2. Overall, our findings suggest that LBP antagonizes cardiomyocyte apoptosis by inhibiting the upregulation of GRK2 induced by I/R injury and saves mitochondrial fission/fusion imbalance and AKT/eNOS signaling. This study may provide new ideas for the study of I/R injury and the rational application of the herbal medicine LBP.

Cardiac GRK2 Protein Levels Show Sexual Dimorphism during Aging and Are Regulated by Ovarian Hormones.

Cardiovascular disease (CVD) risk shows a clear sexual dimorphism with age, with a lower incidence in young women compared to age-matched men. However, this protection is lost after menopause. We demonstrate that sex-biased sensitivity to the development of CVD with age runs in parallel with changes in G protein-coupled receptor kinase 2 (GRK2) protein levels in the murine heart and that mitochondrial fusion markers, related to mitochondrial functionality and cardiac health, inversely correlate with GRK2. Young female mice display lower amounts of cardiac GRK2 protein compared to age-matched males, whereas GRK2 is upregulated with age specifically in female hearts. Such an increase in GRK2 seems to be specific to the cardiac muscle since a different pattern is found in the skeletal muscles of aging females. Changes in the cardiac GRK2 protein do not seem to rely on transcriptional modulation since adrbk1 mRNA does not change with age and no differences are found between sexes. Global changes in proteasomal or autophagic machinery (known regulators of GRK2 dosage) do not seem to correlate with the observed GRK2 dynamics. Interestingly, cardiac GRK2 upregulation in aging females is recapitulated by ovariectomy and can be partially reversed by estrogen supplementation, while this does not occur in the skeletal muscle. Our data indicate an unforeseen role for ovarian hormones in the regulation of GRK2 protein levels in the cardiac muscle which correlates with the sex-dependent dynamics of CVD risk, and might have interesting therapeutic applications, particularly for post-menopausal women.

Degradation of GRK2 and AKT is an early and detrimental event in myocardial ischemia/reperfusion.

BackgroundIdentification of signaling pathways altered at early stages after cardiac ischemia/reperfusion (I/R) is crucial to develop timely therapies aimed at reducing I/R injury. The expression of G protein-coupled receptor kinase 2 (GRK2), a key signaling hub, is up-regulated in the long-term in patients and in experimental models of heart failure. However, whether GRK2 levels change at early time points following myocardial I/R and its functional impact during this period remain to be established.MethodsWe have investigated the temporal changes of GRK2 expression and their potential relationships with the cardioprotective AKT pathway in isolated rat hearts and porcine preclinical models of I/R.FindingsContrary to the maladaptive up-regulation of GRK2 reported at later times after myocardial infarction, successive GRK2 phosphorylation at specific sites during ischemia and early reperfusion elicits GRK2 degradation by the proteasome and calpains, respectively, thus keeping GRK2 levels low during early I/R in rat hearts. Concurrently, I/R promotes decay of the prolyl-isomerase Pin1, a positive regulator of AKT stability, and a marked loss of total AKT protein, resulting in an overall decreased activity of this pro-survival pathway. A similar pattern of concomitant down-modulation of GRK2/AKT/Pin1 protein levels in early I/R was observed in pig hearts. Calpain and proteasome inhibition prevents GRK2/Pin1/AKT degradation, restores bulk AKT pathway activity and attenuates myocardial I/R injury in isolated rat hearts.InterpretationPreventing transient degradation of GRK2 and AKT during early I/R might improve the potential of endogenous cardioprotection mechanisms and of conditioning strategies.

Aldosterone Jeopardizes Myocardial Insulin and β-Adrenergic Receptor Signaling via G Protein-Coupled Receptor Kinase 2.

Hyperaldosteronism alters cardiac function, inducing adverse left ventricle (LV) remodeling either via increased fibrosis deposition, mitochondrial dysfunction, or both. These harmful effects are due, at least in part, to the activation of the G protein-coupled receptor kinase 2 (GRK2). In this context, we have previously reported that this kinase dysregulates both β-adrenergic receptor (βAR) and insulin (Ins) signaling. Yet, whether aldosterone modulates cardiac Ins sensitivity and βAR function remains untested. Nor is it clear whether GRK2 has a role in this modulation, downstream of aldosterone. Here, we show in vitro, in 3T3 cells, that aldosterone impaired insulin signaling, increasing the negative phosphorylation of insulin receptor substrate 1 (ser307pIRS1) and reducing the activity of Akt. Similarly, aldosterone prevented the activation of extracellular signal-regulated kinase (ERK) and the production of cyclic adenosine 3′,5′-monophosphate (cAMP) in response to the β1/β2AR agonist, isoproterenol. Of note, all of these effects were sizably reduced in the presence of GRK2-inhibitor CMPD101. Next, in wild-type (WT) mice undergoing chronic infusion of aldosterone, we observed a marked GRK2 upregulation that was paralleled by a substantial β1AR downregulation and augmented ser307pIRS1 levels. Importantly, in keeping with the current in vitro data, we found that aldosterone effects were wholly abolished in cardiac-specific GRK2-knockout mice. Finally, in WT mice that underwent 4-week myocardial infarction (MI), we observed a substantial deterioration of cardiac function and increased LV dilation and fibrosis deposition. At the molecular level, these effects were associated with a significant upregulation of cardiac GRK2 protein expression, along with a marked β1AR downregulation and increased ser307pIRS1 levels. Treating MI mice with spironolactone prevented adverse aldosterone effects, blocking GRK2 upregulation, and thus leading to a marked reduction in cardiac ser307pIRS1 levels while rescuing β1AR expression. Our study reveals that GRK2 activity is a critical player downstream of the aldosterone signaling pathway; therefore, inhibiting this kinase is an attractive strategy to prevent the cardiac structural disarray and dysfunction that accompany any clinical condition accompanied by hyperaldosteronism.

Calpains mediate isoproterenol-induced hypertrophy through modulation of GRK2.

Inhibition of the Ca2+-dependent proteases calpains attenuates post-infarction remodeling and heart failure. Recent data suggest that calpain activity is elevated in non-ischemic cardiomyopathies and that upregulation of the key cardiac G-protein-coupled receptor kinase 2 (GRK2) signaling hub promotes cardiac hypertrophy. However, the functional interactions between calpains and GRK2 in this context have not been explored. We hypothesized that calpain modulates GRK2 levels in myocardial hypertrophy of non-ischemic cause, and analyzed the mechanisms involved and the potential therapeutic benefit of inhibiting calpain activity in this situation. The oral calpain inhibitor SNJ-1945 was administered daily to male Sprague-Dawley rats or wild-type and hemizygous GRK2 mice treated with 5mg/Kg/day isoproterenol intraperitoneally for 1week. In isoproterenol-treated animals, calpains 1 and 2 were overexpressed in myocardium and correlated with increased calpain activity and ventricular hypertrophy. Oral co-administration of SNJ-1945 attenuated calpain activation and reduced heart hypertrophy as assessed using morphological and biochemical markers. Calpain activation induced by isoproterenol increased GRK2 protein levels, while genetic downregulation of GRK2 expression prevented isoproterenol-mediated hypertrophy independently of calpain inhibition. GRK2 upregulation was associated to calpain-dependent degradation of the GRK2 ubiquitin ligase MDM2 and to enhanced NF-κB-dependent GRK2 gene expression in correlation with calpain-mediated IĸB proteolysis. These results demonstrate that calpain mediates isoproterenol-induced myocardial hypertrophy by modulating GRK2 protein content through mechanisms involving the control of GRK2 stability and expression. Sustained calpain inhibition attenuates isoproterenol-induced myocardial hypertrophy and could be an effective therapeutic strategy to limit ventricular remodeling of non-ischemic origin.

Various effects of AAV9-mediated βARKct gene therapy on the heart in dystrophin-deficient (mdx) mice and δ-sarcoglycan-deficient (Sgcd-/-) mice

So far effective strategies to treat cardiomyopathy in patients with muscular dystrophies are still not clearly defined. Previously, treatment with β-blockers showed beneficial effects on the development of cardiomyopathy in dystrophin-deficient (mdx) mice, but not in δ-sarcoglycan-deficient (Sgcd-/-) mice. We therefore aimed to study a more specific approach to target maladaptive β-adrenergic signalling in these mice. It has been shown that lowering cardiac G-protein-coupled-receptor-kinase-2 (GRK2) activity with βARKct expression, a peptide inhibitor of protein-coupled-receptor-kinase-2 (GRK2), results in improvement of heart failure in several different animal models. We therefore investigated whether adeno-associated virus type 9 (AAV9)-mediated gene delivery of βARKct, could ameliorate cardiac pathology in mdx and Sgcd-/- mice. We found that long-term treatment with AAV9- βARKct-cDNA with a cardiac-specific promoter significantly improves left ventricular systolic function and reduces myocardial hypertrophy in mdx mice, whereas only mild beneficial effects on cardiac function is observed in Sgcd-/- mice. Interestingly, in contrast to mdx mice neither GRK2 nor nuclear-factor-kappaB (NFκB) were upregulated in Sgcd-/- mice. Taken together, effectiveness of AAV-mediated βARKct therapy may vary between different genetic mutations and presumably depend on the state of adrenergic dysregulation mediated through the upregulation of GRK2.

G protein-coupled receptor kinase 2 inhibition improves erectile function through amelioration of endothelial dysfunction and oxidative stress in a rat model of type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is a common cause of erectile dysfunction (ED). It has been demonstrated that G protein-coupled receptor kinase 2 (GRK2) overexpression contributes to diabetic endothelial dysfunction and oxidative stress, which also underlies ED in T2DM. We hypothesized that GRK2 overexpressed and attenuated endothelial function of the cavernosal tissue in a rat model of T2DM. T2DM rats were established by feeding with a high-fat diet (HFD) for 2 weeks and then administering two intraperitoneal (IP) injections of a low dose of streptozotocin (STZ), followed by continuous feeding with a HFD for 6 weeks. GRK2 was inhibited by IP injection of paroxetine, a selective GRK2 inhibitor, after STZ injection. Insulin challenge tests, intracavernous pressure (ICP), GRK2 expression, the protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) pathway, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit gp91phox, nitric oxide (NO), reactive oxygen species (ROS) production, and apoptosis in cavernosal tissue were examined. Less response to insulin injection was observed in T2DM rats 2 weeks after HFD. Markedly increased GRK2 expression, along with impaired Akt/eNOS pathway, reduced NO production, increased gp91phox expression and ROS generation, increased apoptosis and impaired erectile function were found in T2DM rats. Inhibition of GRK2 with paroxetine ameliorated Akt/eNOS signaling, restored NO production, downregulated NADPH oxidase, subsequently inhibited ROS generation and apoptosis, and ultimately preserved erectile function. These results indicated that GRK2 upregulation may be an important mechanism underlying T2DM ED, and GRK2 inhibition may be a potential therapeutic strategy for T2DM ED.

Abstract 318: MicroRNA Regulation of G Protein-Coupled Receptor Kinase 2 After Cardiac Injury

The increase in myocardial expression and activity of G Protein-Coupled Receptor (GPCR) Kinase 2 (GRK2) during the development and progression of heart failure (HF) has been documented to be pathological. Upregulation of GRK2 that is involved with deactivating agonist-activated β-adrenergic receptors (βARs) results in weakened contractile responsiveness through decreases in βAR density, coupling, and sensitivity. Non-GPCR activity of GRK2 has also been shown to be involved in cardiac dysfunction. Studies with knockout or blockade of GRK2 are able to significantly reduce and/or prevent HF phenotypes. Conversely, models with over-expressed GRK2 produce increased injury and accelerated HF development. Being such an important kinase in cardiac disease, regulation of GRK2 expression and activity is critical. Of interest, there is not much known about how GRK2 is regulated (mRNA or protein) in the heart and mechanisms for its up-regulation after cardiac injury are not well understood. There is evidence of GRK2 being regulated by microRNAs (miRNAs) in endothelial and blood cells. We believe cardiac GRK2 may also be regulated in a similar miRNA-mediated fashion. MiRNA profiles become dysregulated with the onset of stress/injury and contribute to orchestrating many classical HF phenotypes, including βAR aberrations. We hypothesize that the expression of a GRK2-regulating miRNA is altered in HF, which contributes to changes in GRK2 regulation resulting in its pathological increase. MiRNA microarray analysis was done on 2-week post-myocardial infarction (MI) murine hearts to examine miRNA profiles compared to sham-operated. Cardiac miRNA species were examined for their expression levels, seed sequence, and TargetScan prediction and alignment to mouse and human GRK2 3’UTR. Candidate miRNAs underwent 3’UTR analysis against GRK2 3’UTR and transfection into AC16 cardiac cells. Two miRNAs were identified to significantly reduce GRK2 protein and/or RNA levels as well directly bind to the 3’UTR of GRK2. Further studies will be done to elucidate the mechanism these miRNAs are able to regulate GRK2 levels. Results from these studies can be applied towards GRK2 inhibition via miRNA-based therapeutics.

Alpha2-adrenoceptors in adrenomedullary chromaffin cells: functional role and pathophysiological implications.

Chromaffin cells from the adrenal medulla participate in stress responses by releasing catecholamines into the bloodstream. Main control of adrenal catecholamine secretion is exerted both neurally (by the splanchnic nerve fibers) and humorally (by corticosteroids, circulating noradrenaline, etc.). It should be noted, however, that secretory products themselves (catecholamines, ATP, opioids, ascorbic acid, chromogranins) could also influence the secretory response in an autocrine/paracrine manner. This form of control is activity-dependent and can be either inhibitory or excitatory. Among the inhibitory influences, it stands out the one mediated by α2-adrenergic autoreceptors activated by released catecholamines. α2-adrenoceptors are G protein-coupled receptors capable to inhibit exocytotic secretion through a direct interaction of Gβγ subunits with voltage-gated Ca2+ channels. Interestingly, upon intense and/or prolonged stimulation, α2-adrenergic receptors become desensitized by the intervention of G protein-coupled receptor kinase 2 (GRK2). In several experimental models of heart failure, there has been reported the up-regulation of GRK2 and the loss of functioning of inhibitory α2-adrenoceptors resulting in enhanced release of adrenomedullary catecholamines. Given the importance of circulating catecholamines in the pathophysiology of heart failure, the recovery of α2-adrenergic modulation of the secretory response from chromaffin cells appears as a novel strategy for a better control of the patients with this cardiac disease.

G protein coupled receptor kinase-2 upregulation causes κ-opioid receptor desensitization in diabetic heart

Activation of κ-opioid receptor (KOR) ameliorates myocardial ischemia/reperfusion (I/R) injury; however, cardioprotective effects of KOR stimulation disappear in type 1 diabetic subjects with hyperglycemia. The molecular mechanisms underlying this phenomenon remain unknown. Here we found that KOR expression was obviously downregulated and KOR agonism-induced contractile-regulatory and cardioprotective effects were significantly impaired in hearts isolated from streptozotocin (STZ) injection-induced diabetic mice. These in vivo data identified cardiac KOR desensitization as a novel characteristic of the diabetic heart. In cultured cardiomyocytes, high glucose (HG) caused obvious KOR downregulation, accompanied by an upregulation of G protein coupled receptor kinase-2 (GRK2). We found that HG exposure increased the interaction between GRK2 and KOR. More importantly, HG-induced KOR downregulation was reversed by small interfering RNA (siRNA)-mediated GRK2 inhibition. GRK2 knockdown also restored KOR agonism-mediated protection against simulated I/R injury in cardiomyocytes. These in vitro data revealed an essential role of GRK2 in HG-induced KOR desensitization. Finally, cardiac-specific GRK2 knockdown by intramyocardial siRNA injection blocked KOR downregulation and restored contractile-regulatory and cardioprotective effects of KOR agonism in hearts of diabetic mice. In conclusion, these data for the first time demonstrate that GRK2 upregulation is largely responsible for cardiac KOR desensitization in diabetic individuals, which provides novel insights into the management of myocardial I/R injury in patients with diabetes.

Regulatory effects of GRK2 on GPCRs and non-GPCRs and possible use as a drug target (Review).

Gprotein-coupled receptor kinase2(GRK2) is a key member of the Gprotein-coupled receptor kinase(GRK) family. GRK2 activity is regulated by the C-terminus of GRK2 which contains a plekstrin homology domain and the N-terminus of GRK2 which contains the RGS homology domain with binding sites for several proteins and lipids such as Gprotein-coupled receptors(GPCRs), Gprotein, phospholipaseC, phosphatidylinositol 4,5-bisphosphate, extracellular signal‑regulated kinase, protein kinaseA and Gβγ. GRK2 phosphorylates the GPCR and enhances the affinity of binding to arrestins, which uncouple the receptors from G proteins, and target the receptors for desensitization and internalization. GRK2 also regulates non‑GPCR desensitization and internalization by phosphorylation, and is important in maintaining the balance between the receptors and signal transduction. Previous findings have indicated that the upregulation of GRK2 in heart failure enhances dysfunctional adrenergic signaling and myocyte death. Collagen-induced arthritis induces the upregulation of GRK2 expression in fibroblast-like synoviocytes. In this review, we discussed the evidence for the association between altered GRK2 levels and various diseases, which suggests that GRK2 may be an effective drug target for preventing and treating heart failure, hypertension and inflammatory disease.

G-Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitors: Current Trends and Future Perspectives.

G-protein-coupled receptor kinase 2 (GRK2) is a G-protein-coupled receptor kinase that is ubiquitously expressed in many tissues and regulates various intracellular mechanisms. The up- or down-regulation of GRK2 correlates with several pathological disorders. GRK2 plays an important role in the maintenance of heart structure and function; thus, this kinase is involved in many cardiovascular diseases. GRK2 up-regulation can worsen cardiac ischemia; furthermore, increased kinase levels occur during the early stages of heart failure and in hypertensive subjects. GRK2 up-regulation can lead to changes in the insulin signaling cascade, which can translate to insulin resistance. Increased GRK2 levels also correlate with the degree of cognitive impairment that is typically observed in Alzheimer's disease. This article reviews the most potent and selective GRK2 inhibitors that have been developed. We focus on their mechanism of action, inhibition profile, and structure-activity relationships to provide insight into the further development of GRK2 inhibitors as drug candidates.

Dexmedetomidine alleviates rat post-ischemia induced allodynia through GRK2 upregulation in superior cervical ganglia.

A transient decrease in G protein-coupled receptor kinase 2 (GRK2) in nociceptors can produce long-lasting neuroplastic changes in nociceptor function, eventually enhancing and prolonging inflammatory hyperalgesia. Here, we investigated the effects of selective α2-adrenoceptor agonist dexmedetomidine (DMED) on GRK2 expression in superior cervical ganglion (SCG) in a rat model of complex regional pain syndrome type I (CRPS-I). The ipsilateral 50% paw withdrawal thresholds (PWTs) to mechanical stimuli decreased significantly starting from 24 h after ischemia-reperfusion (I/R) injury, and lasted for over 3 weeks; the ipsilateral cold allodynia scores, GRK2 protein and mRNA levels in SCGs all increased significantly. No significant differences were found in the contralateral side except GRK2 mRNA reduced significantly after 48 h I/R injury, but still higher than those in the ipsilateral side. Following daily injection of 10 μg/kg of DMED for a maximum of 7 days, the ipsilateral PWTs on days 1, 2, 7, 14, and 21 after DMED administration were significantly higher than those in control group; the GRK2 protein and mRNA expressions in the ipsilateral SCGs were also significantly upregulated; the ipsilateral cold allodynia scores were significantly reduced. No significant differences were found in the contralateral 50%PWTs, cold allodynia scores, and GRK2 protein level except GRK2 mRNA levels increased significantly on days 1 to 7 after DMED administration. Therefore, a transient decrease of GRK2 expression in SCG neurons might be involved in the development and maintenance of allodynia in CRPS-I and DMED might alleviate this allodynia through GRK2 upregulation in SCG neurons.

Downregulation of G protein-coupled receptor kinase 2 levels enhances cardiac insulin sensitivity and switches on cardioprotective gene expression patterns

G protein-coupled receptor kinase 2 (GRK2) has recently emerged as a negative modulator of insulin signaling. GRK2 downregulation improves insulin sensitivity and prevents systemic insulin resistance. Cardiac GRK2 levels are increased in human heart failure, while genetically inhibiting GRK2 leads to cardioprotection in mice. However, the molecular basis underlying the deleterious effects of GRK2 up-regulation and the beneficial effects of its inhibition in the heart are not fully understood. Therefore, we have explored the interconnections among a systemic insulin resistant status, GRK2 dosage and cardiac insulin sensitivity in adult (9month-old) animals. GRK2+/− mice display enhanced cardiac insulin sensitivity and mild heart hypertrophy with preserved systolic function. Cardiac gene expression is reprogrammed in these animals, with increased expression of genes related to physiological hypertrophy, while the expression of genes related to pathological hypertrophy or to diabetes/obesity co-morbidities is repressed. Notably, we find that cardiac GRK2 levels increase in situations where insulin resistance develops, such as in ob/ob mice or after high fat diet feeding. Our data suggest that GRK2 downregulation/inhibition can help maintain cardiac function in the face of co-morbidities such as insulin resistance, diabetes or obesity by sustaining insulin sensitivity and promoting a gene expression reprogramming that confers cardioprotection.

Abstract 208: Mitochondrial-targeted Grk2 Increases Superoxide Formation And Impairs Cardiomyocyte Respiratory Reserve Capacity

β-adrenergic receptors (βARs) are powerful regulators of cardiovascular function and are impaired in heart failure (HF). Signal transduction of βARs is canonically shut down by phosphorylation via G protein-coupled receptor kinase 2 (GRK2) and the subsequent binding of β-arrestins. This process of receptor desensitization is enhanced in HF via the up-regulation of GRK2 and contributes to disease progression. We have recently reported non-canonical actions of GRK2, which contribute to the development of HF independent of βAR desensitization. We have previously shown that GRK2 can act as a pro-death kinase in cardiomyocytes bytranslocating to mitochondria and activating mitochondria permeability transition. This study was designed to gain more understanding of the mitochondrial function of GRK2. We isolated adult cardiomyocytes from cardiac-specific transgenic mice overexpressing GRK2 at levels found in human HF (TgGRK2), and examined superoxide production using the redox sensitive reporter MitoSox Red. Confocal imaging revealed a 4.6 fold increase in superoxide levels in cardiomyocytes overexpressing Grk2 as compared to non-transgenic (NLC) cardiomyocytes (corrected total cell fluorescence 11.59±1.06, TgGRK2 (n= 3 hearts, 88 cells) vs 2.54±0.02 NLC (n=3 hearts, 52 cells), (p&lt;0.001). This indicates that the chronic elevation of GRK2 induces mitochondrial oxidative stress priming the myocyte for enhanced injury. To further explore the mitochondrial actions of GRK2 and consequences of redox stress we examined oxidative phosphorylation by performing oxygen consumption measurements in neonatal rat ventricular myocytes overexpressing GRK2 or GFP-expressing control myocytes. Seahorse analysis showed that cells overexpressing GRK2 have a significant decrease in spare respiratory capacity indicating that cells with elevated GRK2 levels have an impaired capacity to generated ATP during times of stress. Further studies with mutants that limit GRK2 kinase activity or mitochondrial localization demonstrate that mitochondrial GRK2 may be a significant contributor to cellular dysfunction as seen in heart failure.

Improvement of vascular insulin sensitivity by downregulation of GRK2 mediates exercise-induced alleviation of hypertension in spontaneously hypertensive rats

Exercise training lowers blood pressure and is a recommended nonpharmacological strategy and useful adjunctive therapy for hypertensive patients. Studies demonstrate that physical activity attenuates progression of hypertension. However, underlying mechanisms remain elusive. Vascular insulin resistance and endothelial dysfunction plays a critical role in the development of hypertension. The present study investigated whether long-term physical exercise starting during the prehypertensive period prevents the development of hypertension via improving vascular insulin sensitivity. Young (4 wk old) prehypertensive spontaneously hypertensive rats (SHRs) and their normotensive Wistar-Kyoto (WKY) control rats were subjected to a 10-wk free-of-loading swim training session (60 min/day, 5 days/wk). Blood pressure, mesenteric arteriolar vasorelaxation, G protein-coupled receptor kinase-2 (GRK2) expression and activity, and insulin-stimulated Akt/endothelial nitric oxide synthase (eNOS) activation were determined. SHRs had higher systolic blood pressure, systemic insulin resistance, and impaired vasodilator actions of insulin in resistance vessels when compared with WKY rats. Systolic blood pressure in SHRs postexercise was significantly lower than that in sedentary rats. Vascular insulin sensitivity in mesenteric arteries was improved after exercise training as evidenced by an increased vasodilator response to insulin. In addition, exercise downregulated vascular GRK2 expression and activity, which further increased insulin-stimulated vascular Akt/eNOS activation in exercised SHRs. Specific small interfering RNA knockdown of GRK2 in endothelium mimicked the effect of exercise-enhanced vascular insulin sensitivity. Likewise, upregulation of GRK2 by Chariot-mediated delivery opposed exercise-induced vascular insulin sensitization. Taken together, our results suggest that long-term exercise beginning at the prehypertensive stage improves vascular insulin sensitivity via downregulation of vascular GRK2 that may help to limit the progression of hypertension.

G βγ -Independent Recruitment of G-Protein Coupled Receptor Kinase 2 Drives Tumor Necrosis Factor α–Induced Cardiac β-Adrenergic Receptor Dysfunction

Proinflammatory cytokine tumor necrosis factor-α (TNFα) induces β-adrenergic receptor (βAR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. Two different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNFα showed that TNFα alone is sufficient to mediate βAR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling βAR desensitization independent of sympathetic overdrive. TNFα-mediated βAR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in β2AR-overexpressing human embryonic kidney 293 cells showed significant βAR desensitization, GRK2 upregulation, and recruitment to the βAR complex following TNFα. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated βAR phosphorylation and GRK2 recruitment on TNFα. Furthermore, TNFα-mediated βAR phosphorylation was not blocked with βAR antagonist propranolol. Additionally, TNFα administration in transgenic mice with cardiac overexpression of Gβγ-sequestering peptide βARK-ct could not prevent βAR desensitization or cardiac dysfunction showing that GRK2 recruitment to the βAR is Gβγ independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNFα-mediated βAR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNFα-mediated loss in contractility, showing that TNFα-induced βAR desensitization is GRK2 dependent. TNFα-induced βAR desensitization is mediated by GRK2 and is independent of Gβγ, uncovering a hitherto unknown cross-talk between TNFα and βAR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.

Dysfunction of endothelium-dependent relaxation to insulin via PKC-mediated GRK2/Akt activation in aortas of ob/ob mice

In diabetic states, hyperinsulinemia may negatively regulate Akt/endothelial nitric oxide synthase (eNOS) activation. Our main aim was to investigate whether and how insulin might negatively regulate Akt/eNOS activities via G protein-coupled receptor kinase 2 (GRK2) in aortas from ob/ob mice. Endothelium-dependent relaxation was measured in aortic rings from ob/ob mice (a type 2 diabetes model). GRK2, β-arrestin2, and Akt/eNOS signaling-pathway protein levels and activities were mainly assayed by Western blotting. Plasma insulin was significantly elevated in ob/ob mice. Insulin-induced relaxation was significantly decreased in the ob/ob aortas [vs. age-matched control (lean) ones]. The response in ob/ob aortas was enhanced by PKC inhibitor or GRK2 inhibitor. Akt (at Thr(308)) phosphorylation and eNOS (at Ser(1177)) phosphorylation, and also the β-arrestin2 protein level, were markedly decreased in the membrane fraction of insulin-stimulated ob/ob aortas (vs. insulin-stimulated lean ones). These membrane-fraction expressions were enhanced by GRK2 inhibitor and by PKC inhibitor in the ob/ob group but not in the lean group. PKC activity was much greater in ob/ob than in lean aortas. GRK2 protein and activity levels were increased in ob/ob and were greatly reduced by GRK2 inhibitor or PKC inhibitor pretreatment. These results suggest that in the aorta in diabetic mice with hyperinsulinemia an upregulation of GRK2 and a decrease in β-arrestin2 inhibit insulin-induced stimulation of the Akt/eNOS pathway and that GRK2 overactivation may result from an increase in PKC activity.